一种基于抗体结合导致的构象修饰的新型转化生长因子α（TGFα）夹心酶联免疫吸附测定（ELISA）。

A new sandwich enzyme-linked immunosorbent assay (ELISA) for transforming growth factor alpha (TGF alpha) based upon conformational modification by antibody binding.

作者信息

Inagaki H, Katoh M, Kurosawa-Ohsawa K, Tanaka S

机构信息

Laboratory for Biochemistry, Hoechst Japan Limited, Saitama, Japan.

出版信息

J Immunol Methods. 1990 Mar 27;128(1):27-37. doi: 10.1016/0022-1759(90)90460-d.

DOI:10.1016/0022-1759(90)90460-d

PMID:2324504

Abstract

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for human TGF alpha was established utilizing monoclonal and polyclonal anti-synthetic human TGF alpha antibodies with defined epitopes. A polyclonal antibody which was raised in a rabbit and affinity purified by C terminal peptide (hTGF alpha (34-50)) recognized both intact and denatured human TGF alpha. Murine monoclonal antibodies isolated bound only to the denatured form of TGF alpha at the second loop (hTGF alpha (16-33)). However, the rabbit antibody was found to induce a conformation change of intact TGF alpha and the resultant immunocomplex was recognized by monoclonal antibodies. By virtue of this property, the ELISA could detect both native and denatured TGF alpha with the same efficiency with a detection limit of 0.1 ng/ml. Human EGF did not interfere with the ELISA. Production of TGF alpha in several transformed human cell lines was quantitatively examined. Some cell lines were found to secrete TGF alpha, but the production rate was very low, except one melanoma, suggesting that TGF alpha may function only locally in a very limited area in vivo.

摘要

利用具有明确表位的单克隆和多克隆抗合成人转化生长因子α（TGFα）抗体，建立了一种夹心型酶联免疫吸附测定（ELISA）法检测人TGFα。一种在兔体内产生并通过C末端肽（hTGFα(34 - 50)）亲和纯化的多克隆抗体，可识别完整和变性的人TGFα。分离得到的鼠单克隆抗体仅与TGFα在第二个环（hTGFα(16 - 33)）处的变性形式结合。然而，发现兔抗体可诱导完整TGFα的构象改变，且产生的免疫复合物可被单克隆抗体识别。基于这一特性，该ELISA法能够以相同效率检测天然和变性的TGFα，检测限为0.1 ng/ml。人表皮生长因子（EGF）不干扰该ELISA法。对几种转化的人细胞系中TGFα的产生进行了定量检测。发现一些细胞系分泌TGFα，但除一种黑色素瘤外，产生率非常低，这表明TGFα在体内可能仅在非常有限的区域局部发挥作用。